Method for treating diseased or damaged organs

ABSTRACT

A bioremodelable prosthesis for treating a patient with a diseased or damaged organs comprising a first layer that contains acid-extracted fibrillar or non-fibrillar collagen, and a second layer derived from the tunica submucosa of the small intestine that provides structural stability, is pliable and is semi-permeable,pe1 59564443.npc wherein the prosthesis undergoes controlled biodegradation occurring with adequate living cell replacement such that the original prosthesis is replaced by the patient&#39;s living cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/889,079, filed Jul. 7, 1997 now U.S. Pat. No. 6,334,872, which is acontinuation-in-part of U.S. patent application Ser. No. 08/461,756,filed Jun. 5, 1995, now abandoned, which is a continuation of U.S.patent application Ser. No. 08/198,062, filed Feb. 18, 1994, nowabandoned; each of which is in its entirety hereby incorporated byreference.

FIELD OF THE INVENTION

This invention is in the field of tissue engineering. The presentinvention is a resilient, biocompatible two- or three-layered tissuegraft which can be engineered in flat sheets or in tubes with variousluminal diameters and thicknesses. At least one layer is composed ofcollagen or a collagenous material. The present invention is graduallydegraded and bioremodeled by the host's cells which replace theimplanted prosthesis and assume its shape.

BACKGROUND OF THE INVENTION

Each year approximately 300,000 coronary bypass procedures are performedin the United States. The typical treatment for small diameter arteryreplacement has been for surgeons to use the patient's own vessels,usually the saphenous vein from the leg. However, in many cases, the useof the patient's own vessels is not practical because the veins areeither damaged, diseased or are not available. In these cases, syntheticmaterials are used, but with unsatisfactory long-term results. It isstill a continuing goal of researchers to develop prostheses which cansuccessfully be used to replace or repair mammalian tissue, particularlyblood vessels.

SUMMARY OF THE INVENTION

This invention is directed to a method for treating damaged or injuredorgans, particularly blood vessels, by replacing, or repairing, asection of the organ in a patient with a bioremodelable collagen graftprostheses. This prosthesis, when implanted into a mammalian host,undergoes controlled biodegradation accompanied by adequate living cellreplacement, or neo-tissue formation, such that the original implantedprosthesis is bioremodeled by the host's cells before it is digested byhost enzymes. The prosthesis of this invention comprises at least twolayers: (a) at least one layer is composed of collagen or a collagenousmaterial; and (b) at least one layer is composed of material whichprovides structural stability, and is pliable, semi-permeable, andsuturable. In the preferred embodiment of this invention, thetwo-layered prosthesis has an inner (luminal) layer which provides asmooth, thrombosis-resistant flow surface and an outer structural layerwhich provides structural stability, and is pliable, semi-permeable, andsuturable. In another preferred embodiment of this invention theprosthesis has three layers: an inner (luminal) layer which acts as asmooth, thrombosis-resistant flow surface; a middle structural layerwhich provides structural stability, and is pliable, semi-permeable, andsuturable; and, an outer (abluminal) layer. The outer layers of both thetwo-layer or the three-layer prosthesis add strength to the graft andallow the patient's host cells to attach and grow into the graft therebyfacilitating the bioremodeling.

The invention is also directed to methods for preparing bioremodelabletwo-or three-layer tubular blood vessel prostheses by (a) forming atubular structural layer that is pliable, semi-permeable, and suturable;(b) forming an inner layer to act as a smooth flow surface comprisingdeposition of acid-extracted fibrillar collagen onto the luminal surfaceof said structural layer of step (a); and, (c) creating the lumen. Theinner layer may also be treated with drugs for anti-thrombotic effect,such as heparin or other appropriate agent(s). The prosthesis is nextimplanted into a mammalian host where it undergoes controlledbiodegradation accompanied by adequate living cell replacement, orneo-tissue formation, such that the original implanted prosthesis isbioremodeled by the host's cells.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C are schematic cross-sectional view of the preferredprosthesis in accordance with the present invention. FIG. 1A representsa two layer prosthesis with an outer collagenous layer and an innerstructural layer. FIG. 1B represents a prosthesis with three layers:inner and an outer collagenous layers and a middle structural layer.FIG. 1C represents a two layer prosthesis with an inner collagenouslayer and an outer structural layer.

FIG. 2 is a Masson's trichrome stain (10×) a three-layer prosthesis ofthis invention prior to implantation.

FIG. 3 is a Masson's trichrome stain (25×) a three-layer prosthesis ofthis invention prior to implantation.

FIG. 4 is a Masson's trichrome stain (10×) of the proximal anastomosisof a a three-layer prosthesis of this invention implanted as a caninefemoral interposition prosthesis (256 days).

FIG. 5 is a Masson's trichrome stain (10×) of the proximal anastomosisof an e-PTFE graft implanted as a canine femoral interpositionprosthesis (256 days).

FIG. 6 is a Masson's trichrome stain (25×) of the proximal anastomosisof a three-layer prosthesis of this invention implanted as a caninefemoral interposition prosthesis (256 days).

FIG. 7 is a Masson's trichrome stain (25×) of the proximal anastomosisof an e-PTFE graft implanted as a canine femoral interpositionprosthesis (256 days).

FIG. 8 is a Verhoeff's elastic stain (10×) of the proximal anastomosisof a three-layer prosthesis of this invention implanted as a caninefemoral interposition prosthesis (256 days).

FIG. 9 is a Verhoeff's elastic stain (10×) of the proximal anastomosisof an e-PTFE graft implanted as a canine femoral interpositionprosthesis (256 days).

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a method for treating damaged or injuredorgans in a patient with a bioremodelable graft, which, when implantedinto a mammalian host, serves as a functioning replacement for a bodypart, or tissue structure, and will undergo controlled biodegradationoccurring concomitantly with bioremodeling by the host's cells. Inaddition, the prosthesis of this invention, in its various embodiments,thus has dual properties: First, it functions as a substitute body partand second, while still functioning as a substitute body part, itfunctions as a bioremodeling template for the ingrowth of host cells.

When the prosthesis of this invention functions as substitute body part,it is preferably used as a vascular graft. The vascular graft prosthesismay be tubular or flat. Tubular grafts will be used as a conduit tobypass or replace arteries or veins. When formed into flat sheets, theprosthesis can be used as a vascular or intra-cardiac patch. Inaddition, the prosthesis can be implanted to replace diseased or damagedorgans, including the esophagus, intestine, bowel, urethra, andfallopian tubes. These organs all have a basic tubular shape with anouter surface and a luminal surface. Further, the prosthesis can be usedas a conduit for nerve regrowth and regeneration.

The prosthesis of this invention has increased resiliency or“spring-open” or “spring-back” properties. Spring back properties areimportant for applications such as a vascular tubes or patches.

The second function of the prosthesis is that of a template forbioremodeling. “Bioremodeling” is used herein to mean the production ofstructural collagen, vascularization, and epithelialization by theingrowth of host cells at a rate faster than the loss of biomechanicalstrength of the implanted prosthesis due to biodegradation by hostenzymes. The prosthesis retains the distinct characteristics of theoriginally implanted prosthesis while it is remodeled by the body intoall, or substantially all, “self” and as such is functional as afunctioning tissue structure.

The prosthesis is made of at least two layers: (a) at least one layer iscomposed of collagen or a collagenous material that has a smooth,uniform diameter geometry and is non-thrombogenic and (2) at least onelayer which provides structural stability and biomechanical properties.The mechanical integrity means that the prosthesis is non-dilating andnon-aneurysmal during bioremodeling, and additionally is pliable andsuturable. The term “pliable” means good handling properties. The term“suturable” means that the mechanical properties of the layer includesuture retention which permits needles and suture materials to passthrough the prosthesis material at the time of suturing of theprosthesis to sections of natural vessel, a process known asanastomosis. During suturing, such vascular (blood vessel) grafts mustnot tear as a result of the tensile forces applied to them by thesuture, nor should they tear when the suture is knotted. Suitability ofvascular grafts, i.e., the ability of grafts to resist tearing whilebeing sutured, is related to the intrinsic mechanical strength of theprosthesis material, the thickness of the graft, the tension applied tothe suture, and the rate at which the knot is pulled closed.

The prosthesis of this invention is particularly directed to use as abypass or replacement of small diameter blood vessels in the hostpatient. As used herein, and as is understood by those of skill in theart, a small diameter tube is less than 6 mm, typically around 3 to 4mm. A medium diameter tube is between 6 to 12 mm. A large diameter tubeis greater than 12 mm. As an example, the various vascular diametersizes in adult humans are as follows: the diameter of aortic vessels isfrom about 12 to 22 mm; the diameter of the iliac vein is from 8 to 12mm; the diameter of the superficial femoral vein is 6 mm. Above theknee, the femoral is 6 mm; across the knee, the femoral is 4 to 6 mm.

The combination of the two layers of the prosthesis of this inventionwhen used as a tubular vascular graft work advantageously by combining asmooth thrombosis resistant flow surface on the inner (luminal)collagenous layer with the structural layer which, in addition to itsother properties, aids in preventing luminal creep, that is maintainingthe nominal diameter. Dilatation (or aneurysmal) failure occurs when thepulsatile pressure and forces exceed the ability of the graft to resistan increase in diameter. Dilatation or aneurysm formation is an increasein diameter beyond nominal. This occurs in both prosthesis as well as inarteriosclerotic arteries. As used herein, the term “non-dilatating”means that the biomechanical properties of the prosthesis impartdurability so that the diameter of the prosthesis is not stretched,distended, or expanded beyond normal limits after implantation. As isdescribed below, total dilatation of the implanted prosthesis of thisinvention is within acceptable limits. The prosthesis of this inventionacquires a resistance to dilatation as a function of post-implantationcellular bioremodeling by replacement of structural collagen by hostcells at a faster rate than the loss of mechanical strength of theimplanted materials due from biodegradation and remodeling.

Various tubular configurations are embodied by this prosthesis as shownin FIGS. 1A, 1B, and 1C. FIG. 1A shows a two layer prosthesis with anouter collagenous layer and an inner structural layer. FIG. 1B shows aprosthesis with three layers: inner and an outer collagenous layers anda middle structural layer. FIG. 1C shows a two layer prosthesis with aninner collagenous layer and an outer structural layer.

Each of these various embodiments has applicability for particular graftreplacements. The two layer prosthesis shown by FIG. 1A, with an outercollagenous layer and the inner structural layer, is useful as areplacement for vessels or hollow organs which can tolerate a lesssmooth inner or luminal surface, such as the esophagus, intestine,bowel, urethra, or fallopian tubes. The outer collagenous layer addsstrength to the graft and allows the host's cells to attach to it,permitting ingrowth into the graft. In contrast, the prosthesis as shownin FIG. 1B and in FIG. 1C with an inner, smooth collagenous layer areuseful as blood vessel replacements. The inner collagenous layerfunctions as a smooth flow surface.

The structural layer may be made from bioremodelable collagen orcollagenous materials; or biodegradable polymeric materials, such aspolylactic or polyglycolic acid, or combinations thereof; or biostablepolymers, such as polytetrafluoroethylene (PTFE), polyethylene, orcombinations thereof. In the preferred embodiment, collagenous materialfrom collagenous parts of tissue from the mammalian body is used to makethis layer. Such tissue includes but is not limited to intestin, fascialata, or dura mater. The most preferred material for use as a structurallayer is the tunica submucosa layer of the small intestine, termedherein the “intestinal collagen layer.” As used herein, the structurallayer will typically have a thickness of between about 50 microns toabout 150 microns, more preferably between about 75 microns to about 125microns. These dimensions are for an intestinal collagen layer aftermechanical cleaning, but before tubulation by heat welding andcrosslinking, as described below; both mechanical cleaning and heatwelding significantly reduce the “apparent” thickness of the intestinalcollagen layer.

When collagenous material of tissue origin is used to form thestructural layer, it may be crosslinked to provide strength to thestructure. Crosslinking collagenous material also provides somestiffness to the material to improve handling properties. Additionally,crosslinking collagenous material on a mandrel yields a tube of a moreuniform diameter than if the material had not been crosslinked. Thisminimizes the risk of thrombosis which can be enhanced when there isdiscontinuity in the geometry of the vessel. Crosslinking agents shouldbe selected so as to produce a biocompatible material capable of beingbioremodeled by host cells. Various types of crosslinking agents areknown and can be used; this is discussed below with the preferredembodiment. A preferred crosslinking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).There are certain crosslinking agents that cannot be used on theprosthesis of this invention since they will produce a crosslinkedmaterial that will not undergo remodeling by host cells. Glutaraldehyde,for example, is not useful for crosslinking with this invention as theresidual of the glutaraldehyde monomer and lower molecular polymers arecytotoxic. Therefore, it would prevent cell ingrowth and bioremodeling.

The structural layer, at least when made with bioremodelable collagen orcollagenous materials, such as the intestinal collagen layer, will be“semi-permeable,” that is, permitting the ingrowth of host cells forremodeling or for deposition of the collagenous layer, as describedbelow. Crosslinking ICL renders the material relatively less permeableas measured by water porosity testing.

The other layer of the prosthesis is the collagenous layer, the functionof which is to act as a smooth flow surface for whatever its ultimateapplication. When used as the inner, luminal layer of the prosthesis,its function is to provide a smooth contacting surface, particularly ablood contact flow surface.

This smooth collagenous layer may be made from acid-extracted fibrillaror non-fibrillar collagen, which is predominantly type I collagen, butmay also include type 3 or 4 collagen, or both. The collagen used may bederived from any number of mammalian sources, typically bovine, porcine,or ovine skin and tendons. The collagen preferably has been processed byacid extraction to result in a fibril dispersion or gel of high purity.Collagen may be acid-extracted from the collagen source using a weakacid, such as acetic, citric, or formic acid. Once extracted intosolution, the collagen can be salt-precipitated using NaCl andrecovered, using standard techniques such as centrifugation orfiltration. Details of acid extracted collagen are described, forexample, in U.S. Pat. No. 5,106,949, incorporated herein by reference.

Collagen dispersions or gels for use in the present invention aregenerally at a concentration of about 1 to 10 mg/ml, preferably, fromabout 2 to 6 mg/ml, and most preferably at about 2 to 4 mg/ml and at pHof about 2 to 4. A preferred solvent for the collagen is dilute aceticacid, e.g., about 0.05 to 0.1%. Other conventional solvents for collagenmay be used as long as such solvents are compatible.

Additionally, in another embodiment of the invention, the collagenouslayer can include mechanically sheared or chopped collagen fibers. Thechopped collagen fibers improve the spring-back performance of thecollagenous layer. The chopped fibers can be added to the collagensolution used for formation of the acid-extracted collagen gel. Theproperties of the construct incorporating the fibers may be varied byvariations in the length and diameter of the fiber; variations on theproportion of the fiber used, and partially crosslinking fibers. Thelength of the fibers can range from 5 cm to 5.0 cm, and will typicallybe incorporated into the collagen gel at a concentration of 5 to 60.

In another embodiment of the invention, the formation of the inner orouter collagenous layer can incorporate previously formed collagenthreads. For example, a helix, or braid of micron diameter collagenthread could be incorporated as part of the formation of the collageninner layer. The diameter size of the helix or braid of collagen threadcan range from 25 to 50 microns, preferably 25 to 40 microns. Thus, theproperties of the collagen layer can be varied by the geometry of thethread used for the reinforcement. The functionality of the design isdependent on the geometry of the braid or twist. Many of these will alsoeffect the physical properties (i.e, compliance, radial strength, kinkresistance, suture retention). Physical properties of the thread mayalso be varied by crosslinking.

Some portion or all of the fibers used could be polylactic acid. Thephysical and degradation properties of the lactic acid fibers themselvescan be manipulated by varying the molecular weight, as well as the useof the D or L racemes or a mixture of D/L forms of lactic acid. Otherfibers fabricated from degradable polymers could also be used, such aspolyglycolic acid, caprolacatone, and polydioxinone.

Small Diameter Two-Layer Tubular Prosthesis

Method of Preparation

To further describe the prosthesis of this invention, the process ofmaking a small diameter two layered tubular prosthesis will be describedin detail below. The described two-layered prosthesis has an inner(luminal) surface composed of acid-extracted fibrillar collagen and theouter (abluminal) structural layer composed of mammalian tunicasubmucosa from the small intestine. Flat prosthesis can be similarlyprepared with the described methods by using a flat form instead of amandrel to produce the prosthesis.

1. Preparation of the Structural Layer.

The submucosa, or the intestinal collagen layer, from a mammaliansource, typically pigs, cows, or sheep, is mechanically cleaned bysqueezing the raw material between opposing rollers to remove themuscular layers (tunica muscularis) and the mucosa (tunica mucosa). Thetunica submucosa of the small intestine is harder and stiffer than thesurrounding tissue, and the rollers squeeze the softer components fromthe submucosa. As the mechanically cleaned submucosa may have somehidden, visibly nonapparent damage that affects the consistency of themechanical properties, the submucosa may be chemically cleaned to removesubstances other than collagen, for example, by soaking in buffersolutions at 4° C., without the use of any detergents such as Triton orSDS, or by soaking with NaQH or trypsin, or by other known cleaningtechniques.

After cleaning, the intestinal collagen layer (ICL) should besterilized, preferably with the use of dilute peracetic acid solutionsas described in U.S. Pat. No. 5,460,962, incorporated herein byreference. Other sterilization systems for use with collagen are knownin the art and can be used.

The ICL may be tubulated by various alternative means or combinationsthereof. The ICL material may be formed into a tube in either the normalor the everted position, but the everted position is preferred. The tubemay be made mechanically by suturing, using alternating knot stitcheswith suitable suture material. The knot stitch is advantageous as itallows the tube to be trimmed and shaped by the surgeon at the time ofimplantation without unraveling. Other processes to seam the submucosamay include adhesive bonding, such as the use of fibrin-based glues orindustrial-type adhesives such as polyurethane, vinyl acetate orpolyepoxy. Heat bonding techniques may also be used including heatwelding or laser welding of the seam, followed by quenching, to seal thesides of the thus formed tube. Other mechanical means are possible, suchas using pop rivets or staples. With these tubulation techniques, theends of the sides may be either butt ended or overlapped. If the sidesare overlapped, the seam may be trimmed once the tube is formed. Inaddition, these tubulation techniques are typically done on a mandrel soas to determine the desired diameter.

The thus formed structural tube can be kept on a mandrel or othersuitable spindle for further processing. To control the biodegradationrates and therefore the rate of prosthesis strength decrease duringbioremodeling, the prosthesis is preferably crosslinked, using asuitable crosslinking agent, such as (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Crosslinking the prosthesis also aidsin preventing luminal creep, in keeping the tube diameter uniform, andin increasing the burst strength. It is believed that crosslinking theintestinal collagen layer also improves the suture retention strength byimproving resistance to crack propagation.

2. Deposition of Collagenous Layer(s).

Bovine collagen may be deposited on the internal surface of thesubmucosa as described in Example 5 of U.S. Pat. No. 5,256,418,incorporated herein by reference. Briefly, the structural intestinalcollagen layer is sealed at one end by luer fittings and the collagendispersion fills the tube. This step may also be accomplished asdescribed in the above-referenced patent application using a hydrostaticpressure head. The inner layer of collagen can also be deposited byflowing collagen into both ends of the tube simultaneously. The tube isthen placed into a bath of 20% polyethylene glycol (PEG) in isotonicphosphate buffered saline (PBS), pH about 7. The osmotic gradientbetween the internal collagen solution and outer PEG solution incombination cause a simultaneous concentration and deposition of thecollagen along the lumen of the internal structural layer wall. The tubeis then removed from the PEG bath, and a glass rod with a diameterdesired diameter of the prosthesis lumen is inserted into the collagensolution. The prosthesis is then allowed to dry. The tube is thenrehydrated in PBS. This process allows the collagenous layer to fillslight irregularities in the intestinal structural layer, thus resultingin a prosthesis of uniform thickness. The procedure also facilitates thebonding of the collagen gel to the intestinal collagen layer. Acollagenous layer of varying thickness and density can be produced bychanging the deposition conditions which can be determined by routineparameter changes. The same procedures can be used to apply the collagento the outer surface of the submucosa to create a three-layerprosthesis.

3. Treatment of the Inner Collagenous Layer.

The prosthesis construct is thrombogenic in small diameter blood vesselreplacements. It can only be used in vascular applications in high flow(large diameter) vessels. Therefore, the prosthesis must be renderednon-thrombogenic to be useful for small diameter blood vessel repair orreplacement.

Heparin can be applied to the prosthesis, by a variety of well-knowntechniques. For illustration, heparin can be applied to the prosthesisin the following three ways. First, benzalkonium heparin (BA-Hep)solution can be applied to the prosthesis by dipping the prosthesis inthe solution and then air-drying it. This procedure treats the collagenwith an ionically bound BA-Hep complex. Second, EDC can be used toactivate the heparin, then to covalently bond the heparin to thecollagen fiber. Third, EDC can be used to activate the collagen, thencovalently bond protamine to the collagen and then ionically bondheparin to the protamine. Many other heparin coating, bonding, andattachment procedures are well known in the art which could also beused.

Treatment of the inner layer with drugs in addition to heparin may beaccomplished. The drugs may include for example, growth factors topromote vascularization and epitheliazation, such as macrophage derivedgrowth factor (MDGF), platelet derived growth factor (PDGF), endothelialcell derived growth factor (ECDGF); antibiotics to fight any potentialinfection from the surgery implant; or nerve growth factors incorporatedinto the inner collagenous layer when the prosthesis is used as aconduit for nerve regeneration. The treatment of the abluminal (outer)layer may also be done in a manner similar to that for the luminal(inner layer).

4. Cell Ingrowth Facilitation.

If the structural layer is made of ICL which is crosslinked thecompleted two or three layer prosthesis can be laser drilled to createmicron sized pores through the completed prosthesis for aid in cellingrowth using an excimer laser at either KrF or XeF wavelengths. Thepore size can vary from 20 to 100 microns, but is preferably from about30 to 60 microns and spacing can vary, but about 500 microns on centeris preferred.

5. Sterilization.

The completed graft is then sterilized. The preferred method is to useperacetic acid as described in U.S. Pat. No. 5,460,962, incorporatedherein by reference. Sterilization may also be accomplished bysubjecting the prosthesis to a gamma radiation treatment (⁶⁰Co) of 10.0to 25.0 kGy. The radiation dose eliminates all microorganisms withoutadversely affecting the biomechanical properties of the prosthesis.

Prosthesis Test Standards

Various tests, analysis and performance parameters have been developedover the years for vascular graft prosthesis and can be used by those ofskill in the art to evaluate the prosthesis characteristics. Thesemethods are detailed in Abbott et al., “Evaluation and performancestandards for arterial prostheses,”Journal of Vascular Surgery, Volume17, pages 746-756 (1993) and “American National Standard for VascularGraft Prostheses,” American National Standards Institute (1986).

Methods of Treating a Patient with a Prosthesis

For indications where the patient has diseased or damaged arteries orveins, replacement or bypass of a section of vessel with a vasculargraft or prosthesis is necessary. An exemplary bypass procedure, acoronary artery bypass graft operation, sometimes referred to as CABG(“cabbage”), is done to reroute, or “bypass”, blood around cloggedarteries and improve the supply of oxygenated blood to the heart. Bloodflow through these arteries is often narrowed or obstructed byaccumulation of fat, cholesterol and other substances. This narrowing istermed “atherosclerosis” which can sometimes lead to a coronary arrest(heart attack).

The prosthesis of the present invention can be used to serve as aready-to-use vascular graft for such indications that requirereplacement or bypass to both restore physical function and eventually,because of its bioremodelable characteristics, its biological function.

In the situation of a coronary bypass, the heart is made accessible byopening the body cavity. The vascular prosthesis is attached at one endto the aorta and the other end to the coronary artery below the blockedarea. In the situation of a vessel replacement, the vessel is madeaccessible and is ligated at two points located at either end of thesection to be replaced so as to prevent the flow of blood through thesection. The prosthesis is engrafted at or near the points where theoriginal vessel is removed.

Surgical placement of the graft, in both the bypass and replacementsituations, can be accomplished in either of two ways. Aninterpositional placement of the graft is end-to-end anastomosis whereinthe section of vessel is completely severed and one end of the graft issutured to the end of the vessel. End-to-side anastomosis, commonly donein bypass procedures, is when an incision is made in the sidewall of thevessel to create an opening and one end of the graft is sutured to theopening. The placement of the ends of the graft may be both end-to-end,both end-to-side or a combination of the two techniques.

Once both ends of the graft are sutured in place, blood flow is resumedand monitored as the graft functions as a prosthetic blood vessel.Further, as the prosthesis of the present invention is bioremodelable,it functions as a remodeling template for the ingrowth of host cells.Over time, the graft is replaced with the patient's cells which, at thesame time, degrade the graft and replace it with new matrix so that itbecomes a new tissue structure.

The following examples further describe the materials and methods usedin carrying out the invention. The examples are not intended to limitthe invention in any manner.

EXAMPLES Example 1 Two and Three Layer Tubular Prosthesis

The small intestine of a pig was harvested and mechanically stripped sothat the tunica submucosa is delaminated from the tunica muscularis andthe luminal portion of the tunica mucosa of the section of smallintestine. (The machine was a striper, crusher machine for themechanical removal of the mucosa and muscularis layers from thesubmucosal layers using a combination of mechanical action (crushing)and washing using hot water.) This was accomplished by running theintact intestine through a series of rollers that strip away successivelayers. The intestinal layer was machine cleaned so that the submucosalayer solely remains. The submucosa was decontaminated or sterilizedwith 0.1% peracetic acid for 18 hours at 4° C. and then washed after theperacetic acid treatment.

This machine cleaned intestinal collagen layer (ICL) was mounted andstretched on a frame so that it was under slight tension both radiallyand longitudinally. Coarse running basting stitches (6-0 Novafil®) wereapplied to form a small diameter tube, with the submucosa in the evertedposition. The stretched ICL tissue was cut in half to overlap and formflaps. A fine seam through both layers of ICL was formed using 6-0Novafil® suture with an alternating knot stitch so that a final internaldiameter of 4.5 mm was obtained. The flaps were then removed. Thesmall-diameter ICL tube, used as the structural layer, was placed onto a4.5 mm glass rod and crosslinked with 100 mmol EDC (Pierce) for 18 hoursat room temperature.

The machine cleaned intestinal collagen layer (ICL) in a fully hydratedcondition was mounted and wrapped on a mandrel so that the endsoverlapped. The ICL wrapped mandrel was heated to 62° C. plus or minus10° C. for 15 minutes in a moist atmosphere, followed by quenching at 4°C. in iced aqueous solution for 5 minutes. The tubulated ICL was thencrosslinked with EDC for 6 to 18 hours, rinsed, and removed from themandrel.

Polycarbonate barbs (luer lock fittings that are funnel shaped on oneend) were placed tightly in either end of the tube and then the tube wasplaced horizontally in a deposition fixture. A 15 ml reservoir of 2.5mg/ml acid-extracted fibrillar collagen, termed “dense fibrillarcollagen” (“DFC”) (U.S. Pat. No. 5,256,418, incorporated herein byreference) with a hydrostatic pressure head of 150 mmHg (for 5 feet) wasattached via the barbs. (The pressure will depend on the height of thecollagen reservoir.) The collagen was allowed to fill the lumen of theICL tube and was then placed into a stirring bath of 20% MW 8000polyethylene glycol (Sigma Chemical Co.) for 16 hours at 4° C. Theapparatus was thee dismantled. To fix the luminal diameter, a 4 mmdiameter glass rod was placed into the collagen-filled ICL tube. Theprosthesis was then allowed to dry for 18 hours at 4° C.

A layer of acid extracted fibrillar collagen was deposited onto a 4.0 mmdiameter porous ceramic mandrel as described in Example 4, U.S. Pat. No.5,256,418, incorporated herein by reference, for 6 hours and dehydratedat 4° C.

The ICL tube, as described above, was placed over the dried collagen anda second layer of dense fibrillar collagen (DFC), as described above,was applied for 18 hours to the outside (abluminal) of the ICL.

Pores were drilled in the ICL/DFC or the DFC/ICL/DFC using an excimerlaser at either KrF or XeF wavelengths. The pore size was about 50microns and spacing was 500 microns on center.

The construct was rehydrated in 4° C. 1M PBS for 6 hours. The prosthesiswas treated with application of benzalkonium heparin in isopropranol.Sterilization was accomplished with 0.1% peracetic acid for 18 hours at4° C.

The prosthesis was packaged and sterilized with 10.0 to 25.0 kGy ofgamma radiation (⁶⁰Co). (The prosthesis can also be shipped dry andrehydrated in saline, prior to implantation.)

Example 2 Remodeling of the Collagen Graft Long Term Implant Histology

Three-layer prosthesis were implanted in the infra-renal aorta ofrabbits using standard surgical techniques. Proline, 7-0, was used toconstruct end to end anastomoses to the adjacent arteries. The graftswere 1.5 cm in length and 3 mm in diameter. No anti-platelet medicationswere administered post-operatively.

Following pressure perfusion with McDowells-Trump fixative, the graftswere explanted, and submitted for light and electron microscopy.Specimens from 30, 60, 90, 120, and 180 day implants were available.Materials were examined with H/E, VonGieson elastica, Masson'sTrichrome, g-Actin, Factor VIII, and Ram-11 (macrophage) stains, andpolarized microscopy. Qualitative morphometric comparisons were made tostained non-implanted retention samples.

Histological evaluations demonstrated that the graft was readily invadedby host cells. The luminal collagen was resorbed and remodeled with theproduction of new collagen by host myofibroblasts. The ICL was readilyinvaded, re-populated by host cells, and remodeled. Endothelial cellswere demonstrated on the lumninal surface of the prosthesis.

At 30 days, large numbers of mononuclear inflammatory cells were seen onboth the luminal and abluminal surfaces of the collagen. Modest numbersof Ram-11 positively staining macrophages, were observed. At the surfaceof the cast collagen, there was cell mediated collagen resporption andremodeling. There was minimal loss of the collagen bulk at this time.

At 60 days, the cellular response was more myofibroblastic thaninflammatory. Significant amounts of new collagen as well as smallamounts of elastin were readily identified. About 50 percent of the castcollagen had been remodeled. Endothelial cells as identified by SEMappearance, TEM (Weibel-Palade bodies) and positive Factor VIIIstaining, covered the surface of the remodeling construct.

At 90 days, the matrix surrounding the myofibroblasts (as identifiedwith g-actin) stained prominently for collagen. The cytoplasm of cellsthemselves had reduced amounts of cytoplasm as compared to previoustimepoints.

At 120 days, the stroma demonstrated well organized predominantlyradially and longitudinally oriented myofibroblasts and host producedcollagen. Significant amounts of elastin could be identified. Greaterthan 90 percent of the implanted collagen had been remodeled. No Ram-11staining macrophages were identified.

At 180 days, cells and the matrix of the neo-artery were quite mature.The cells were small with minimal cytoplasm. The collagen was dense anddistinctly radially and longitudinally oriented.

There was no histological evidence of an immune response to either theluminal collagen layer or the abluminal ICL layer. No grafts becamedilated or aneurysmal.

Example 3 Comparison of Three-Layer Prosthesis and e-PTFE

Both two-layer and three-layer small diameter prosthesis were implantedand evaluated over time for patency and remodeling.

FIGS. 4-9 show the results of a comparison of three-layer prothesis witha similarly configured contra-lateral reference material, e-PTFE, in acanine femoral artery study. The grafts were implanted in canines asfemoral interposition prosthesis. Grafts were explanted from 30 to 256days.

Histological evaluation of the three-layer collagen graft demonstratedcellular ingrowth into the graft at 30 days, with more than 90 percentof the graft collagen remodeled by 90 days; and a mature ‘neo-artery’ at180 days. Host tissue bridged the anastomosis by 60 days with theanastomosis only demarcated by the non-resorbable sutures. Thepredominant cell type in the neo-artery was a positive g-Actin stainingsmooth muscle like cell. The surface of the remodeled graft was lined byendothelial cells as demonstrated by SEM, TEM and Factor VIII staining.

In contrast, at times to 256 days, no ingrowth into the e-PTFE arterywas observed either across the anastomosis or along the body of thegraft. Only a thin smooth muscle cell hyperplastic response wasdemonstrated extending from the adjacent artery a short distance on thegraft's luminal surface. The graft was encapsulated by mature fibroustissue with no evidence of cellular or tissue extension into the graft.

FIG. 4 is a Masson's trichrome stain (10×) of the proximal anastomosisof the three-layer prosthesis at 256 days compared with FIG. 5 of ane-PTFE graft. FIG. 6 is also a Masson's trichrome stain at 25× of theproximal anastomosis of a three-layer prosthesis at 256 days comparedwith FIG. 7 of an e-PTFE graft.

FIG. 8 is a Verhoeff's elastic stain (10×) of the proximal anastomosisof a three-layer prosthesis implanted as a canine femoral interpositionprosthesis at 256 days compared with FIG. 9 of an e-PTFE graft.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one skilled in the art that certainchanges and modifications may be practiced within the scope of theinvention, as limited only by the scope of the appended claims.

1. A bioremodelable prosthesis for treating a patient with a diseased ordamaged organ, comprising a first layer that contains acid-extractedfibrillar or non-fibrillar collagen, and a second layer derived from thetunica submucosa of the small intestine that provides structuralstability, is pliable, and is semi-permeable, wherein said prosthesisall undergoes controlled biodegradation occurring with adequate livingcell replacement such that the original prosthesis is replaced by thepatient's living cells.
 2. The prosthesis of claim 1, wherein theprosthesis is tubular and the diseased or damaged organ is an artery ora vein.
 3. The prosthesis of claim 2, wherein the tubular prosthesis hasa diameter of less than 6 mm.
 4. The prosthesis of claim 2, wherein thetubular prosthesis has a diameter of between 6 to 12 mm.
 5. Theprosthesis of claim 2, wherein the tubular prosthesis has a diameter ofgreater than 12 mm.
 6. The prosthesis of claim 1, wherein the diseasedor damaged organ is the esophagus, intestine, bowel, urethra, orfallopian tubes.
 7. The prosthesis of claim 1, wherein said first layerhas a smooth flow surface.
 8. The prosthesis of claim 7, wherein thesmooth flow surface is thrombosis-resistant.
 9. The prosthesis of claim1, wherein said second layer is crosslinked.
 10. The prosthesis of claim9, wherein said second layer is crosslinked with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). 11.The prosthesis of claim 1, wherein said second layer has a thickness ofbetween about 50 microns to about 150 microns.
 12. The prosthesis ofclaim 1, wherein the tunica submucosa of the small intestine ismechanically cleaned to remove the tunica muscularis and the tunicamucosa.
 13. The prosthesis of claim 12, wherein said mechanicallycleaned tunica submucosa is chemically cleaned.
 14. The prosthesis ofclaim 1, wherein the tunica submucosa of the small intestine issterilized.
 15. The prosthesis of claim 1, wherein said prosthesisfurther comprises a third layer.
 16. The prosthesis of claim 1, whereinthe first layer is on the inside of the prosthesis.
 17. The prosthesisof claim 1, wherein the second layer is on the inside of the prosthesis.18. The prosthesis of claim 1, wherein the prosthesis is treated with adrug.
 19. The prosthesis of claim 1, wherein the first layer is treatedwith a drug.
 20. The prosthesis of claim 1, wherein the second layer istreated with a drug.
 21. The prosthesis of claim 18, 19, or 20, whereinthe drug is heparin.
 22. The prosthesis of claim 18, 19, or 20, whereinthe drug is a growth factor.